WO2009155711A1 - Analyse des molécules d'interférence « signal » la-5 de lactobacillus acidophilus - Google Patents

Analyse des molécules d'interférence « signal » la-5 de lactobacillus acidophilus Download PDF

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WO2009155711A1
WO2009155711A1 PCT/CA2009/000901 CA2009000901W WO2009155711A1 WO 2009155711 A1 WO2009155711 A1 WO 2009155711A1 CA 2009000901 W CA2009000901 W CA 2009000901W WO 2009155711 A1 WO2009155711 A1 WO 2009155711A1
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Prior art keywords
bifidobacterium
composition
molecules
probiotic
lactobacillus
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PCT/CA2009/000901
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English (en)
Inventor
Mansel Griffiths
Maira Medellin-Pena
Veronique Delcenserie
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University Of Guelph
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Priority to CA2765298A priority Critical patent/CA2765298A1/fr
Priority to EP21212833.4A priority patent/EP4019529A1/fr
Priority to EP09768681.0A priority patent/EP2307444B1/fr
Priority to US13/001,328 priority patent/US20110262400A1/en
Priority to ES09768681T priority patent/ES2908705T3/es
Publication of WO2009155711A1 publication Critical patent/WO2009155711A1/fr
Priority to US15/696,255 priority patent/US10687543B2/en
Priority to US16/800,698 priority patent/US20200178567A1/en
Priority to US18/168,163 priority patent/US20230226139A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to the control of pathogenic bacteria in mammals. More particularly, the invention relates to the isolation and identification of molecules secreted/derived from probiotic bacteria for use in compositions and methods for the treatment and/or prevention of infection by harmful pathogenic bacteria.
  • the isolated molecules are useful in nutritional and medical food products which provide probiotics to the gastrointestinal tract of a mammal.
  • Enterohaemorrhagic Escherichia coli O157:H7 is a member of the attaching and effacing Escherichia coli (AEEC) (3) that form specific structures known as attaching and effacing (AE) lesions in the host intestinal epithelial wall, which allow EHEC 0157 to intimately attach to the epithelial membrane in order to achieve colonization (18, 22, 24).
  • AE lesion formation initial attachment of the bacterium is followed by the injection of bacterial proteins into the host cell (8, 17, 21) through a specialized translocation apparatus, termed a type III secretion system (TTSS). This results in the cytoskeletal rearrangement and effacement of the microvilli. Finally a 94-kDa bacterial outer membrane protein, termed intimin is required (19), resulting in the formation of the bacterium-host cell pedestal structure (9, 10, 27, 37).
  • TTSS type III secretion system
  • EHEC enteric bacteria
  • enteric microbes A number of enteric bacteria, including EHEC, are known to produce and/or respond to chemical signals called autoinducers. The use of this cell-to-cell signaling mechanism facilitates enteric microbes to regulate important traits that allow them to successfully colonize and/or start infection in their host (20).
  • EHEC virulence- specific genes are regulated by quorum sensing (QS) (34, 35) mediated by the autoinducer-3/epinephrine/norepinephrine signaling system (36).
  • QS quorum sensing
  • Autoinducer-3 (Al- 3) is a molecule produced by the commensal gastrointestinal microbiota that seems to resemble the hormones epinephrine and norepinephrine produced by the host (36) and is believed to allow the enteric pathogens to organize a concerted activation/repression of specifically required genes.
  • an EHEC sensor kinase, QseC which binds AI-3 and the hormones epinephrine/norepinephrine and regulates virulence in a rabbit infection model provides evidence that this QS system participates in interkingdom cross-communication (5).
  • enteric pathogens possess an extremely complex regulatory system that is used to systematically compete in such a challenging environment and inhibition of this QS system may lead to an attenuation of virulence.
  • Salmonella spp. are widespread with in the environment. S. Typhimurium DT104 typically is resistant to the antibiotics ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (R-type ACSSuT)(48). Salmonella enterica serovar Typhimurium requires the expression of the TTSS for a number of important virulence factors like bacterial invasion, macrophage apoptosis and enteropathogenesis (41, 43, 44, 46 and 47). TTSS gene transcription is activated in response to environmental signals (39, 40 and 45). Cattle are thought to be a primary reservoir through which salmonella multi-resistant pathogens can enter the food supply.
  • the human gastrointestinal tract harbors a complex microbial ecosystem containing a large number and variety of bacteria that has a major impact on gastrointestinal function and thereby on human health and well-being.
  • some opportunistic bacteria are considered to be detrimental and cause adverse conditions such as diarrhea, infections, gastroenteritis and endotoxaemia, while other bacteria are considered "probiotic", in that they perform beneficial functions for the human organism (49).
  • Probiotic bacteria are known to stimulate the immune system and exert a competitive exclusion of pathogenic and putrefactive bacteria, reduce the amounts of ammonia and cholesterol in the blood, and promote absorption of minerals (50). Additionally, probiotic bacteria produce antagonist effects against pathogenic microorganisms; stimulate the immune system; improve lactose digestion; are lipolytic, thereby allowing fats to be more digestible; reduce plasma cholesterol; protect the intestinal mucosa, thereby assuring effective assimilation of the nutritive substances; produce polysaccharides that are active on some tumors; and reduce viability of some enzyme-producing microorganisms which catalyze the conversion of procarcinogenic substances into carcinogenic substances. It is believed that the probiotic bacteria exert their effects in a synergistic manner to curtail and retard the growth of pathogenic and detrimental bacteria of the gut (51 and 52).
  • US 20040161422 discloses a nutritional food product comprising at least one probiotic bacteria to improve gut function.
  • U.S. 20040115177 discloses methods of administering probiotic bacteria to livestock animals in an amount effective to reduce the amount of hazardous bacteria.
  • Dietary supplements such as those for example sold as part of the PARINAT tm line is formulated with Lactobacillus acidophilus strain L.B. and is stated to be beneficial for general digestive and intestinal problems.
  • L. acidophilus La-5 may affect virulence-related gene expression in Escherichia coli 0157:1-17 (29).
  • La-5 cell spent medium was used and found to affect bacterial transcriptional regulators, however, the studies were all conducted in vitro on Escherichia coli cultures and thus the conclusions could not support or identify the bacterial factor(s) responsible for the regulation of the EHEC 0157 OS system. It was thus concluded in the study that animal models were required to characterize the efficacy and potential use of the L. acidophilus La-5 in mammalian therapeutic embodiments.
  • the invention provides novel molecules secreted/derived from probiotic bacteria.
  • the novel molecules are secreted/derived from probiotic bacteria meaning that they are secreted by probiotic bacteria directly into the medium or derived from culture fractions.
  • the invention thus provides isolated probiotic proteinaceous fractions from probiotic bacteria and also provides the novel molecules from isolated from such fractions.
  • the invention describes the isolation, characterization and methods of use of the molecules of the invention to prevent or treat infection by harmful bacteria as an alternative or adjunct to traditional antibiotic therapy.
  • the molecules of the invention can be used ingested to improve health and incorporated into beverage and food sources to improve nutritional qualities.
  • the molecules of the invention are low molecular weight and in aspects, proteinaceous as well as heat-stable and partially affected by enzymatic treatment.
  • the secreted molecules of the present invention can also be used as a nutritional supplement to help maintain and/or increase the general health of a mammal and may be incorporated into a variety of food and beverage products for ease of ingestion as well as incorporated into medicaments.
  • the secreted molecules of the present invention can be regarded in one aspect as probiotic.
  • probiotic it is generally defined as a live microbial food supplement which beneficially affects the host human or animal by improving its intestinal microbial balance.
  • probiotic is meant to encompass the secreted molecules from probiotic bacteria.
  • secreted molecules from probiotic bacteria, said secreted molecules being effective in vitro and in vivo to prevent and/or treat bacterial infection.
  • compositions comprising lyophilized probiotic proteinaceous fractions as effective in the prevention and/or treatment of infection by harmful bacteria. Such lyophillized fractions may also be used as a source of mammalian nutritional health.
  • the present invention are isolated secreted molecules from a probiotic bacteria selected from Lactobacillus, Bifidobacteria and Streptococcus.
  • the Bifidobacteria is a species selected from Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis Bifidobacterium crudilactis.
  • the probiotic bacteria is a Lactobacillus selected from Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus.
  • the bacterium is Lactococcus Lactis.
  • the probiotic bacteria is from a Streptococcus such as Streptococcus thermophilus.
  • the secreted molecules of the present invention are effective for treatment and prophylactic therapy against infectious bacteria such as but not limited to EHEC O157:H7 and Salmonella enterica.
  • composition comprising one or more secreted molecules from a probiotic bacterium, said composition being effective to reduce and/or prevent harmful bacterial infection in mammals.
  • composition comprising one or more secreted molecules from a probiotic bacterium and an antibiotic, said composition being effective to reduce and/or prevent harmful bacterial infection in mammals.
  • composition comprising one or more secreted molecules from a probiotic bacterium, a sugar source and optionally an antibiotic, said composition being effective to reduce and/or prevent harmful bacterial infection in mammals.
  • the sugar source comprises glucose.
  • compositions comprising one or more secreted molecules from a bacteria selected from Lactobacillus, Bifidobacterium and Streptococcus and mixtures thereof.
  • the secreted molecules are proteinaceous.
  • the secreted molecules are small, low molecular weight peptides.
  • the secreted molecules can withstand heating at up to about 90 0 C, freezing, thawing, lyophilization and/or spray drying.
  • a secreted molecule from Lactobacillus acidophilus comprises one of the following sequences: YPVEPF, YPPGGP, YPPG and NQPY.
  • composition comprising a secreted molecule from Lactobacillus acidophilus (La-5), wherein said molecule can inhibit colonization by EHEC 0157:1-17 in vivo in a mammal.
  • La-5 Lactobacillus acidophilus
  • composition comprising a secreted molecule from Lactobacillus acidophilus (La-5), wherein said molecule comprises one of the following amino acid sequences: YPVEPF, YPPGGP, YPPG and NQPY and said molecule can prevent and/or treat infection of EHEC O157:H7.
  • the amino acid sequences may have substitutions that do not adversely affect the activity of the secreted molecule.
  • composition comprising a secreted molecule from a Bifidobacterium selected from Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis and/or Bifidobacterium crudilactis, wherein said composition can prevent and/or treat infection of EHEC O157.H7 in vivo in a mammal.
  • a food product, beverage product, medicament or nutritional supplement that comprises one or more secreted molecules of the present invention from a bacterium selected from Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactis, Streptococcus thermophilus and combinations thereof.
  • a bacterium selected from Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactis, Streptococc
  • an ingestible health product for mammals wherein said ingestible health product has probiotic characteristics and comprises one or more secreted molecules from Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis and/or Bifidobacterium crudilactis.
  • Lactobacillus acidophilus La-5
  • Lactobacillus fermentum Lactobacillus rhamnosus
  • Lactococcus Lactis Lactococcus Lactis
  • Streptococcus thermophilus Streptococcus thermophilus
  • the present invention is a method for preventing and/or therapeutically treating infections by Escherichia coli O157:H7 and/or Salmonela, the method comprising administering to a subject an effective amount of a composition comprising one or more secreted molecules from Lactobacillus acidophilus (La-5).
  • the secreted molecules may further comprise those from Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactis, Streptococcus thermophilus and combinations thereof.
  • the present invention also provides a method for preventing the carriage by a food production animal of Salmonella strains that cause human salmonellosis.
  • the method comprises the step of administering an effective amount of secreted molecules from probiotic bacteria to the food production animal prior to exposure to Salmonella strains that cause human salmonellosis.
  • the administration of the secreted molecules from probiotic bacteria is accomplished by feeding a feed supplement or additive which comprises an effective amount of said secreted molecules, or by supplying a water treatment additive or inoculum to the animals' drinking water.
  • the invention therefore provides a feed supplement composition comprising secreted molecules from probiotic bacteria and a water additive comprising secreted molecules from probiotic bacteria.
  • the probiotic bacteria may be selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactis, Streptococcus thermophilus and combinations thereof.
  • Lactobacillus acidophilus La-5
  • Lactobacillus fermentum Lactobacillus rhamnosus
  • Lactococcus Lactis Lactococcus Lactis
  • Streptococcus thermophilus Bifidobacterium longum
  • Bifidobacterium bifidum Bifidobacterium infantis
  • Streptococcus thermophilus and combinations thereof.
  • a method of preventing infection by harmful bacteria in a mammal comprising administration of an effective amount of a secreted molecule(s) from a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium crudilactis, Streptococcus thermophilus and combinations thereof.
  • a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum, Bifidobacterium
  • According to another aspect of the present invention is a method of preventing colonization by harmful bacteria in a mammal, the method comprising administration of an effective amount of a secreted molecule(s) from a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum and Bifidobacterium infantis and/or Bifidobacterium crudilactis.
  • a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum and Bifido
  • a method of improving the general health of a mammal comprising administration of an effective amount of a secreted molecule(s) from a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum and Bifidobacterium infantis and/or Bifidobacterium crudilactis.
  • a probiotic bacteria selected from the group consisting of Lactobacillus acidophilus (La-5), Lactobacillus fermentum, Lactobacillus rhamnosus, Lactococcus Lactis, Streptococcus thermophilus, Bifidobacterium Iongum, Bifidobacterium bifidum and Bifidobacterium infantis and/or Bifidobacterium cru
  • the molecule(s) of the invention may be provided isolated and/or purified or within a cell free culture fraction from the probiotic bacteria.
  • the secreted molecules may be provided within a composition, edible food product or supplement or ingestible liquid. They can be used in conjunction with whole probiotic bacteria and with pharmaceuticals such as known antibiotics.
  • FIG. 1 Fluorescent micrographs of HEp-2 cells incubated for 6 h with EHEC strain 43984. Bright fluorescence with the fluorescein isothiocyanate-phalloidin stain, indicating aggregation of foci of alpha-actinin underneath adherent EHEC (arrows) was visualized under the microcolonies by fluorescence microscopy.
  • A Infected cells
  • B Non-infected cells
  • C EHEC infected cells co-incubated with 40 ⁇ l of peptide fraction of L acidophilus La5
  • D EHEC LuxS (-ve) infected cells.
  • FIG. 1 Bioluminescence images from 10 8 CFU EHEC 0157 infected mice. Images were obtained on the 3 rd , 5 th , and 7 th day post-infection. Areas in which luminescent EHEC 0157 is present are shown as color-overlay.
  • Figure 5 Effect of LA-5 cell-free spent medium fraction (F54) and Bifidobacteria CFSM on the induction of hilA in Salmonella Typhimurium via LuxS assay. Standard deviations from the mean are calculated from 2 independent trials with 3 wells per trial. Expression of hilA gene is monitored by luminescence (RLU) produced by the Salmonella construct.
  • RLU luminescence
  • Figure 6 Effect of LA-5 and Bifidobacteria CFSM and CFSM fractions (F54) on the induction of LEEl in Enterohaemorrhagic E. coli O157:H7 via LuxS assay. Standard deviations from the mean are calculated from 2 independent trials with 3 wells per trial. Expression of LEEl is monitored by luminescence (RLU) produced by the E. coli O157:H7 construct.
  • Figure 7. Effect of LA-5 and Bifidobacteria CFSM (CFSM fraction 54 [F54] separated by cation exchange chromatography [CEX]) on the induction of LEEl in Enterohaemorrhagic E. coli O157:H7 via LuxS assay. Standard deviations from the mean are calculated from 2 independent trials with 3 wells per trial. Expression of LEEl is monitored by luminescence (RLU) produced by the E. coli O157:H7 construct.
  • FIG. 8 Luminescence activity of LEElr.luxCDABE and LEE2::luxCDABE fusions in E. coli O157.H7 (C3, C4) grown in LB broth alone (EHEC/LB) or in LB broth supplemented with 10% of L acidophilus La-5 CFSM fractions 25 to 40 (EHEC/F). Data was collected after 16 h growth. Results were expressed as relative light units (RLU) defined as counts min * and adjusted to OD 6 oo (RLU/OD 6 oo)- The data are mean ⁇ SD values of three independent replicates.
  • RLU relative light units
  • FIG. 9 Autoinducer-2 bioassay conducted three times with the same samples.
  • EHEC O157:H7 (ATCC 43894) was grown for 16 h in LB broth alone (EHEC/LB) or supplemented with 10% of L acidophilus La-5 CFSM fractions 25 to 40 (EHEC/F). The cell-free supernatants from these cultures were collected as described in the methods section. Results were expressed as relative light units (RLU) defined as counts min "1 and adjusted to OD 6 oo (RLU/OD 6 oo)- The data are mean ⁇ SD values of three independent replicates.
  • RLU relative light units
  • FIG. 14 E. coli O157:H7 construct C3 (LEEl::/ux) and C4 (LEE2::/ux) grown in LB broth supplemented with medium conditioned by the growth of probiotic LAB. Constructs grown in LB:MRS broth were used as positive controls (data not shown). Light induction is reported as relative light units (RLU) per cell.
  • RLU relative light units
  • FIG. 15 f. coli O157:H7 construct Cl (LEEl;;lux), C2 (LEE2::lux), C3 (LEEl::/ux) and C4 (LEE2::/ux) grown in LB broth supplemented with medium conditioned by the growth of probiotic LAB. Constructs grown in LB:MRS broth were used as positive controls (data not shown). Light induction is reported as relative light units (RLU) per cell.
  • EHEC O157:H7 strain 43894 was grown in LB broth supplemented with CFSM of probiotic LAB. Positive and negative controls were V. harveyi strain BB152 (+) and E.
  • the present invention provides secreted molecules isolated from probiotic bacteria and further culture fractions of the bacteria that can minimize, inhibit and treat infection by harmful enteric pathogens in mammals.
  • the molecules are demonstrated to be effective both in vitro and in vivo.
  • the molecule(s) have been isolated and characterized from Lactobacillus acidophilus (La-5) as well as from strains of Bifidobacterium such as but not limited to Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis and Bifidobacterium crudilactis (Delcenserie, V., F. Gavini, H. Beerens, O. Tresse, C. Franssen, and G. Daube. 2007.
  • 'secreted/derived' is meant that the probiotic bacteria secrete the novel molecules directly into the culture medium.
  • the molecules can also be formed indirectly within the culture medium.
  • the novel secreted molecules of the invention in aspects are small peptides that are temperature resistant (can be heated, frozen and thawed and still exhibit activity), are stable for long periods of time frozen (over two years), can be produced readily in large volumes (for example about 2mg/L), can be lyophilized and spray dried.
  • the molecules can be incorporated into a variety of substances for administration to a mammal such as any type of animal and humans.
  • the secreted molecules can be incorporated into any type of food product, nutritional supplement or beverage for animal or human consumption.
  • the secreted molecules of the invention can be administered in a manner to an animal or human for the effective treatment of bacterial infection such as by EHEC 0157:1-17 or Salmonella.
  • the treatment can be in conjunction with other antibiotics or other therapies as is desired.
  • the secreted molecules of the invention can be used in compositions and in methods in addition to use of whole probiotic bacteria.
  • the secreted molecules are isolated from Lactobacillus acidophilus (La-5), wherein said molecule comprises one or more of the following amino acid sequences: YPVEPF, YPPGGP, YPPG and NQPY. It is understood by one of skill in the art that these sequences can be altered by deletion, substitution or insertion so long as the activity of the secreted molecules is not substantially affected to reduce and/or prevent bacterial infection.
  • the sequences can further have insertions, substitutions, or deletions of one or more of the amino acid residues.
  • the molecules of the invention may further be altered with glycosylation, unglycosylation, organic and inorganic salts and covalently modified.
  • molecules modified to increase in vivo half life e.g., PEGylated.
  • Possible but non limiting modifications to the molecules of the invention include modifications comprising combinations of amino acid substitutions together with a deletion of one or more amino acids or the addition of one or more amino acids.
  • the molecules of the invention can be provided in a therapeutically effective amount alone or within a composition and may vary according to factors such as the infection state/health, age, sex, and weight of the recipient.
  • Dosage periods may be adjusted to provide the optimum therapeutic response and may be at the discretion of the attending physician or veterinarian. For example, several divided doses may be administered daily or on at periodic intervals, and/or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the amount of the molecule for administration will depend on the route of administration, time of administration and varied in accordance with individual subject responses. Suitable administration routes are intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal administration.
  • Compositions comprising the molecules or the culture fractions of the invention may comprise about 0.1% to about 90% by weight of the active and any range there-in-between.
  • the molecules or culture fractions may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the infection being treated, whether a recurrence of the infection is considered likely, or to prevent infection etc.
  • the administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc.
  • the administration may be intermittent, e.g., the molecules may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable.
  • the compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids.
  • suitable pH a suitable pH and/or be iso-osmotic with physiological fluids.
  • compositions according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
  • a composition containing the secreted molecules in an acceptable carrier is administered to an animal at least about three weeks prior to shipment of the animal in an amount effective to reduce the amount of Salmonella in the animal both before and after harvest.
  • the secreted molecules from probiotic bacteria may be delivered in an acceptable carrier via a food route of administration (e.g., milk product, water, feed, or any suitable medium) or by a medicinal route of administration (e.g., oral or intranasal innoculation).
  • Acceptable carriers for the secreted molecules from probiotic bacteria include feed products for the livestock animal, including, for example, milk or yogurt cultures.
  • a dry form of the secreted molecules from probiotic culture can also be produced and added to feed by the process of lyophilization.
  • Lyophillized secreted molecules may be delivered to animals by any suitable route of administration including via dry feed and water. By administering such therapy in advance of transport, significant levels of hazardous bacteria such as Salmonella, are reduced in the livestock pre and post slaughter.
  • administration of the isolated secreted molecules from probiotic bacteria can be accomplished by any method likely to introduce the molecules into the digestive tract.
  • the bacteria can be mixed with a carrier and applied to liquid or solid feed or to drinking water.
  • the carrier material should be non-toxic to the animal.
  • the molecules can also be formulated into a composition provided as an inoculant paste to be directly injected into an animal's mouth.
  • the formulation can include added ingredients to improve palatability, improve shelf-life, impart nutritional benefits, and the like. If a reproducible and measured dose is desired, the molecules can be administered by a rumen cannula, as described herein.
  • the amount of the secreted molecules isolated from probiotic bacteria to be administered is governed by factors affecting efficacy.
  • the secreted molecules from one or more strains of probiotic bacteria can be administered together.
  • a combination of strains can be advantageous because individual animals may differ as to the strain which is most persistent in a given individual.
  • the secreted molecules from probiotic bacteria can be administered as a preventive, to prevent animals not presently carrying E. coli O157:H7 from acquiring the strain by exposure to other animals or environments where E. coli O157:H7 is present. Young calves and mature animals about to be transferred to a new location, such as a feed lot, are attractive candidates for preventive administration.
  • Treatment of animals carrying E. coli 0157:1-17 can be accomplished to reduce or eliminate the amount of E. coli O157:H7 carried by the animals, by administering the secreted molecules from probiotic bacteria to E. coli O157:H7 infected animals. Animals known to be shedding E. coli O157:H7 in feces, or those raised where E. coli O157:H7 is known to exist are suitable candidates for treatment with the molecules of the invention.
  • the methods for administering the secreted molecules from probiotic bacteria are essentially the same, whether for prevention or treatment. Therefore, the need to first determine whether E. coli O157:H7 being carried by the animals is removed. By routinely administering an effective dose to all the animals of a herd, the risk of contamination by E. coli O157:H7 can be substantially reduced or eliminated by a combination of prevention and treatment.
  • compositions of the novel molecules of the invention whether isolated or in isolated culture fraction can also be used in conjunction (formulated with) with a sugar source such as for example glucose in amounts of up to about 0.01% to about 0.1% or more by weight of the composition.
  • compositions of the invention may be presented in dosage forms such as in a capsule.
  • amounts of the active isolated molecule will vary depending on the particular food or beverage and may contain any amount up to about 100% of the product, especially when formulated as an ingestible capsule. It is also understood by one of skill in the art that the molecules of the invention whether isolated or provided as within a culture fraction can be combined with the use of probiotic bacteria in methods of treatment or for nutritional supplementation.
  • Lactobacillus acidophilus La-5 CFSM decreased E. coli O157:H7 attachment to tissue culture cells.
  • L acidophilus La-5 SM influenced EHEC 0157 T3SS (29). Down-regulation of important virulence-related gene expression was presently detected after EHEC 0157 was grown in medium supplemented with biologically active fractions of L acidophilus La-5 CFSM (La-5 fractions) when compared with EHEC 0157 grown in the same medium without the addition of La-5 fractions.
  • La-5 fractions biologically active fractions of L acidophilus La-5 CFSM
  • the addition of La-5 fraction would have an influence on EHEC 0157 adhesion to eukaryotic cells in vitro and in vivo. Adhesion and AE lesion formation in eukaryotic cells (HEp-2 and HeLa cell lines, respectively) were substantially reduced when La-5 fractions were added before exposure to E.
  • Adherence of EHEC to human epithelial cells involves the activation of the adhesin intimin, an outer membrane protein encoded by the eae gene (9, 26, 27, 37).
  • Previous work (27) showed that production of intimin-specific antisera blocked adherence of EHEC to HEp-2 cells.
  • the immunogenic capacity of intimin has been extensively studied in order to develop anti-EHEC and anti-EPEC vaccines (6, 7, 12, 28). The results demonstrate that secreted molecules from probiotic bacteria could be used to prevent EHEC adherence to epithelial cells in tissue culture models.
  • EHEC 0157 bioluminescent imaging of bacterial colonization on SPF ICR mice was determined. Five different cell concentrations, ranging from 10 s to 10 9 cells per dose, were used for a single challenge with EHEC 0157 bioluminescent strain. The bioluminescent signal for mice infected with 10 5 cells was very weak throughout the experiment and only at an inoculation of 10 7 CFU or greater was the signal strong enough to be visualized and computed (Table 3). Based on previous work in which EHEC 0157 proliferated in mice intestines within 24 h of infection (2), it was expected that a dose of 10 s CFU would have been enough to emit a strong light output. The aim was to monitor EHEC 0157 colonization in vivo in a short period of time, an inoculation dose of 10 8 CFU was selected for the challenge studies.
  • La-5 biologically active fraction reduces attachment of EHEC to intestinal epithelium of ICR mice.
  • EHEC 0157 was recovered from the feces of all groups of mice that were infected with the organism (i.e. groups 2, 3 and 4) throughout the study.
  • mice from group 4 were showing signs of dehydration and physical deterioration and were re-evaluated every 8 h (Fig. 4).
  • Three mice from group 4 died within the evaluation period and the rest showed a significant reduction in body temperature ( ⁇ 34 5C).
  • the end point of group 4 was reached and the remaining mice were euthanized (Table 4).
  • Bioluminescent signals from mice in groups 2, 3 and 4 were taken and analyzed in order to compare their light intensities at the specified times.
  • On the third day of the experiment all mice were orally infected with 10 8 CFU EHEC 0157. Bioluminescence was monitored on the third, fifth and seventh day after infection.
  • CFSM CFSM fractionated by size exclusion chromatography
  • SEC size exclusion chromatography
  • coli 0157:1-17 was further characterized as follows to identify further stains of probiotic bacteria effective against EHEC: (1) The activation or repression of LEE operons was monitored using EHEC strain (ATCC 43894) transformed with gene reporter constructs containing luciferase gene luxCDABE (kindly provided by Dr. Haifeng Wang). These constructs operate under the transcriptional control of the LEE promoters. The expression of LEE operons was measured as light emission produced by E. coli O157:H7 constructs after exposure to medium conditioned by the growth of the probiotic strains.
  • Extracellular fractions from ⁇ . infantis cultures were studied after 24 h growth. After centrifugation (6000 g, 10 min) of 1 litre of culture, the supernatant was filtered through cellulose acetate membrane filters (pore size: 0.22 ⁇ m). The cell-free spent medium (CFSM) was then concentrated via lyophilisation to 1/100 of the original volume. The lyophilized CFSM was resuspended in molecular Biology grade water and separated by size exclusion chromatography (SEC) and the active fractions were stored at -20 9 C for further analysis. Ion exchange chromatography (IEC) was used following the SEC since this is suitable for sample fractionation, purification and screening.
  • IEC Ion exchange chromatography
  • the different fractions (basic and acidic proteins concentrated by IEC and their flow-troughs) were then analyzed with the LuxS assay to determine which one of these fractions possesses the desired activity. After we confirm the presence of active molecules the fractions will be used in multidimensional analysis, such as 2-D gel electrophoresis and HPLC. At the same time we will carry out tissue culture assays with Salmonella ente ⁇ ca serovar Typhimurium and possibly other foodborne pathogens.
  • the first step of separation of molecules from bulk quantities was performed by using size exclusion chromatography (SEC). Following the separated fractions, EHEC O157:H7 bioassays were performed to confirm the presence of the biologically active molecule(s). Consequently, analysis of the biologically active fractions was carried out by electrospray mass spectroscopy (ES-MS) and nuclear magnetic resonance (NMR). Results from the ES-MS and NMR showed that the biologically active fractions were still excessively complex, evading a conclusive report of the nature of the studied fractions. Biologically active fractions were subjected to pH sensitivity, enzymatic and temperature treatments in order to try to shed some light in their nature.
  • SEC size exclusion chromatography
  • the total protein content of the reconstituted CFSM was quantified using the BioRad DC protein assay kit Il (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Freeze-dried CFSM was stored at -20 ? C prior to the assays.
  • CFSM Five millilitres of CFSM were directly deposited on a P2 Biogel (Bio-Rad, Missasauga, ON., Canada) column (exclusion, 100 to 1,800Da; 2.5 xlOO cm; Bio-Rad Laboratories Ltd.) and run at room temperature in 18- ⁇ water at a gravity flow rate of 0.8 ml/min, and eighty 5 ml fractions were collected.
  • the fractions collected were freeze-dried and resuspended in ImI 18- ⁇ water for preliminary screening against EHEC LEEl, LEE2 and AI-2 production as previously described (29).
  • the total protein content of the fractions was quantified using the BioRad DC protein assay kit II. Fractions showing a strong inhibitory activity against LEE expression and AI-2 production were selected.
  • L acidophilus strain La-5 was grown under anaerobic conditions at 37°C in mMRS medium (29).
  • E. coli O157:H7 strain VS94 (36) was grown in Luria-Bertani broth (LB) (BD Diagnostic Systems).
  • the bioluminescent strain of E. coli O157:H7 (luxCDABE) was grown in LB agar supplemented with ampicillin (Amp) and kanamycin (Km) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) at a concentration each of 50 ⁇ g/ml and incubated overnight at 37°C.
  • a single colony was taken from the plate and subcultured in LB broth and high glucose Dulbecco's minimum essential medium (DMEM/High) (Sigma-Aldrich Canada Ltd.) supplemented with the antibiotics and incubated overnight at 37 g C on a shaker at 150 rpm.
  • DMEM/High high glucose Dulbecco's minimum essential medium
  • the correlation between luminescence and cell count in LB broth was established by a standard plate count technique and by the measurement of the bioluminescence for 1 ml of culture serial dilutions with a tube luminometer (MGM Instruments, Hamden, CT).
  • MGM Instruments, Hamden, CT tube luminometer
  • HeLa human cervix adenocarcinoma epithelial cells
  • EMEM complete Eagle's minimal essential medium
  • FBS fetal bovine serum
  • L. acidophilus CFSM (F33 and F34) were added to treatment group wells.
  • a negative control for AE lesion formation we used an E. coli O157:H7 /uxS-negative strain.
  • the negative control group wells were inoculated with 10 5 E. coli O157:H7 strain VS94 with or without supplementation with 100 ⁇ M propanolol, and with only the selected fractions of L acidophilus.
  • Propanolol was used to suppress complementation of the AE phenotype by the hormones epinephrine and norepinephrine produced by the eukaryotic cells.
  • EHEC 0157 strain 43894 After inoculation of EHEC 0157 strain 43894 into treatment and positive control wells, the slides were incubated for 6 h at 37°C in the presence of 5% CO 2 . The cells were then washed three times with phosphate-buffered saline (PBS) and fresh medium was added. Cells were incubated for another 3 h and then washed six times with PBS and fixed in 4% paraformaldehyde. Fixed and washed cells were permeabilized by treating slides with 0.1% Triton X-100 in PBS for 15 min. Cells were incubated with 0.2% bovine serum albumin (BSA) (Invitrogen Canada Inc.) in PBS for 1 h.
  • BSA bovine serum albumin
  • HEp-2 epithelial cells were a kind gift from Dr. Carlton Gyles (Department of Pathobiology, University of Guelph). Briefly, HEp-2 cells grown in EMEM supplemented with 10% (v/v) FBS were plated onto 24-well tissue culture plates at 2 x 10 5 cells ml 1 and incubated for 24 h in the presence of 5 % CO 2 . The cells were then maintained during the assay in serum and antibiotic-free EMEM.
  • Washed cells were lysed with 0.1 % Triton X-100. Released bacteria present in the suspension were collected and appropriate dilutions were plated on LB agar. To evaluate if the percentage of adherence in the treatment groups was significantly different from that of the control group, where the recovered counts from the control group (2.2 X 10 7 CFU ml "1 ) were considered to be 100%, the percentage of adherence in the negative control and treatment groups were calculated using the following equation.
  • mice SPF female ICR mice were obtained at 3 weeks of age from Taconic Farms (Hudson, NY), and used for the experiments after one-week acclimation. Mice were housed at the Isolation Unit of the Central Animal Facility (University of Guelph) in a temperature controlled environment with a 12 h light/dark cycle. Animal care was provided in accordance with the animal utilization protocol no. 04R030 (University of Guelph) and the Guide to the Care and Use of Experimental Animals (1). Mice were fed sterilized solid rodent chow and water. When needed, water was supplemented with Amp and Km at a concentration of 400 mg L "1 and 200 mg L "1 , respectively. Each mouse was assessed daily for weight, body temperature, signs of dehydration, posture and alertness.
  • Bioluminescent imaging was performed as previously described (4) with minor modifications. Briefly, bioluminescent imaging was monitored on the 3 rd , 5 th and 7 th day after infection. Prior to imaging, mice were anesthetized with a cocktail composed of ketamine (60 mg kg "1 ) and medetomidine (0.75 mg kg "1 ). Atipamezole (2.25 mg kg "1 ) was used to reverse the effects of the anesthetics. All drugs were administered intraperitoneal ⁇ . Both bioluminescent and photo images of mice were taken with a cooled slow-scan CCD camera (NightOWL Molecular Imager, EG&G Berthold Technologies, Wildbad, Germany). The integration time for bioluminescence was one minute at low resolution.
  • mice were euthanized with carbon dioxide (CO 2 ).
  • mice Fresh feces of mice were weighed and suspended in PBS (0.5 g of feces per 4.5 ml of 0.1% [w/v] sterile peptone water) to obtain a concentration of 100 mg ml "1 .
  • the fecal suspensions were serially diluted 10-fold and appropriate dilutions were plated in triplicate on LB agar alone and on LB agar supplemented with 50 ⁇ g ml "1 Amp and Km. Colonies that developed after incubation for 24 h at 37?C were counted.
  • the limit of detection was 10 2 CFU g "1 feces.
  • a value of 10 2 g ⁇ feces was assigned to any culture showing no detectable colonies for the purpose of statistical analysis.
  • the effect of temperature on the activity of the CFSM was tested by heating from 30 0 C to 100°C, with increments of 10°C for a period of 20 min. All treated CFSM were tested for inhibitory activity using the EHEC O157:H7 constructs and the autoinducer bioassay described previously herein.
  • Enzymes (0.1 mg ml " ): Proteinase K, pepsin +
  • the absorbance was recorded at 214 and 280 nm by means of a UV detector (SpectraSYSTEM, ThermoFinnigan, Thermolnstruments Inc.).
  • the eluted peaks were pooled, freeze- dried and concentrated 10 times in 18- ⁇ water.
  • the column was calibrated with ⁇ - lactalbumin standard (2.0 mg ml "1 ).
  • the calibration curve was used to determine the average molecular weight of the unknown samples.
  • Chromatographic graphics were obtained using the Chromatography Workstation ChromQuestTM. Total protein content of the collected peaks was measured as described previously (Table 5.1).
  • ⁇ -lactalbumin (MW of 14.2 kDa) was eluted at min 9.3 while the elution of the peptide peaks started at 23 min.
  • Peptide peaks collected were concentrated and desalted before being sent for LC-MS analysis and peptide sequencing.
  • Mass spectrometry was carried out using an Agilent HPLC, coupled to an Agilent 6110 single quadripole LC/MS (Agilent Technologies).
  • the molar masses of three peptide peaks (Fl, Fill and FIV) were detected at m/z 994, 997, 1019, 1078, 1139, 1289 and 2466.
  • Peptide peak (FII) showed no signal peaks (Figs. 11, 12 and 13).
  • the peptide sequencing analysis of peaks Fl, FII and FIV showed that the peptide fractions are composed of 4 to 6 amino acid residues (Table 10).
  • the amino acid sequences obtained are partial peptide sequences of larger peptides or small proteins due to possible blocked N-termini.
  • Blocked N-termini provide the single largest impediment to protein sequence analysis. An estimated 50-80% of all proteins naturally have chemically modified N-termini.
  • the sequential Edman analysis sequences the N- terminal and internal protein.
  • the N-terminal amino acid is reacted with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) protein.
  • PITC phenylthiocarbamyl
  • TZA trifluoroacetic acid
  • ATZ anilinothiazolinone
  • the intermediate is converted to the more stable phenylthiohydantoin (PTH) amino acid derivative and subsequently separated by HPLC, compared against a standard, and identified by the sequencer software.
  • BLAST analysis of the peptide sequences The amino acid sequences of the peptide peaks were introduce in the Basic Local Alignment Search Tool (BLAST) and found a number of matches. BLASTp was done using default opening and gap penalties and a default scoring matrix. We will mention only the 100% homology (Table 5.4).
  • Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli Proceedings of the National Academy of Science, USA 96:15196-15201 Sperandio, V., A. G. Torres, J. A. Gir ⁇ n, and J. B. Kaper. 2001 Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli 0157 H7 Journal of Bacteriology 183:5187-5197 Sperandio, V., A. G. Torres, B. Jarvis, J. P. Nataro, and J. B. Kaper.

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Abstract

Cette invention concerne des molécules sécrétées isolées et caractérisées provenant de bactéries probiotiques du genre Lactobacillus, Lactococcus, Streptococcus ou Bifidobacterium destinées à être utilisées dans des compositions et des procédés pour traiter et/ou prévenir l'infection par des bactéries pathogènes nocives telles que Salmonella ou E. coli. Les molécules sécrétées isolées peuvent également être utilisées dans des produits alimentaires à visée nutritionnelle ou médicale qui fournissent des probiotiques à l'appareil gastro-intestinal d'un mammifère.
PCT/CA2009/000901 2008-06-27 2009-06-26 Analyse des molécules d'interférence « signal » la-5 de lactobacillus acidophilus WO2009155711A1 (fr)

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CA2765298A CA2765298A1 (fr) 2008-06-27 2009-06-26 Molecules bacteriennes probiotiques et leur utilisation dans des methodes pour traiter ou prevenir une infection causee par des bacteriesnefastes et pour contribuer a la sante nutritionnelle
EP21212833.4A EP4019529A1 (fr) 2008-06-27 2009-06-26 Molécules bactériennes probiotiques et leur utilisation dans des procédés pour traiter/prévenir une infection causée par des bactéries néfastes et pour contribuer à la santé nutritionnelle
EP09768681.0A EP2307444B1 (fr) 2008-06-27 2009-06-26 Analyse des molécules d'interférence du signal de lactobacillus acidophilus la-5
US13/001,328 US20110262400A1 (en) 2008-06-27 2009-06-26 Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health
ES09768681T ES2908705T3 (es) 2008-06-27 2009-06-26 Análisis de moléculas de interferencia de la señal de lactobacillus acidophilus La-5
US15/696,255 US10687543B2 (en) 2008-06-27 2017-09-06 Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health
US16/800,698 US20200178567A1 (en) 2008-06-27 2020-02-25 Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health
US18/168,163 US20230226139A1 (en) 2008-06-27 2023-02-13 Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health

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US20150044188A1 (en) * 2013-08-12 2015-02-12 Mansel Griffiths Antiviral methods and compositions comprising probiotic bacterial molecules
WO2015181534A1 (fr) * 2014-05-29 2015-12-03 The University Of Manchester Lysat antibactérien de bactéries probiotiques
CN107814840A (zh) * 2017-12-12 2018-03-20 浙江辉肽生命健康科技有限公司 一种生物活性多肽pkypvepf及其制备方法和应用
WO2018165765A1 (fr) * 2017-03-16 2018-09-20 Microsintesis Inc. Molécules probiotiques pour réduire la virulence d'agents pathogènes
WO2018165764A1 (fr) * 2017-03-16 2018-09-20 Microsintesis Inc. Compositions et procédés faisant intervenir des molécules probiotiques
US10687543B2 (en) 2008-06-27 2020-06-23 University Of Guelph Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health
CN112175872A (zh) * 2020-10-12 2021-01-05 中科美大(福建)生物科技有限公司 一种鼠李糖乳杆菌及其制剂、应用
WO2021234102A1 (fr) 2020-05-20 2021-11-25 Nolivade Procédé de décontamination
EP3993819A4 (fr) * 2019-07-02 2023-09-13 Microsintesis Inc. Inhibiteurs de détection du quorum et/ou métabolites post-biotiques et méthodes associées

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US20230226139A1 (en) 2023-07-20
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US20110262400A1 (en) 2011-10-27
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